Abstract
The synaptonemal complex is a tripartite proteinaceous ultrastructure that forms between homologous chromosomes during prophase I of meiosis in the majority of eukaryotes. It is characterized by the coordinated installation of transverse filament proteins between two lateral elements and is required for wild-type levels of crossing over and meiotic progression. We have generated null mutants of the duplicated Arabidopsis transverse filament genes zyp1a and zyp1b using a combination of T-DNA insertional mutants and targeted CRISPR/Cas mutagenesis. Cytological and genetic analysis of the zyp1 null mutants reveals loss of the obligate chiasma, an increase in recombination map length by 1.3- to 1.7-fold and a virtual absence of cross-over (CO) interference, determined by a significant increase in the number of double COs. At diplotene, the numbers of HEI10 foci, a marker for Class I interference-sensitive COs, are twofold greater in the zyp1 mutant compared to wild type. The increase in recombination in zyp1 does not appear to be due to the Class II interference-insensitive COs as chiasmata were reduced by ∼52% in msh5/zyp1 compared to msh5 These data suggest that ZYP1 limits the formation of closely spaced Class I COs in Arabidopsis Our data indicate that installation of ZYP1 occurs at ASY1-labeled axial bridges and that loss of the protein disrupts progressive coalignment of the chromosome axes.
Highlights
The synaptonemal complex (SC) is a proteinaceous ultrastructure that forms between homologous chromosomes during midprophase I of meiosis and plays a critical role in coordinating the repair of programmed DNA doublestrand breaks (DSBs) to form cross-over (CO) products [1, 2]
Studies have shown that the transverse filaments (TFs) are oriented such that the C termini are associated with lateral elements potentially interacting with DNA, while the N-terminal domains localize to the central region [2, 24]
In the zyp1 null mutants, the rare occurrence of univalent chromosomes indicated a CO control defect leading to loss of CO assurance, consistent with previous observations in ZYP1RNAi knockdown lines [14]
Summary
Plant Materials. zyp1a-1 (SALK_040213) [14]; zyp1a-2 (CRISPR/Cas derived 14 bp deletion in exon 3 c.298_311 del GATGAGAAGCTTTG); zyp1a-3 (CRISPR/ Cas derived 11 bp deletion in exon 3 c.301_311 del GAGAAGCTTTG); zyp1b-1 (SALK_050581) [14]; zyp1b-1 (CRISPR/Cas derived 1 bp insertion in exon 1 c.60_61 ins T); asy (SALK_046272) [41]; asy (SALK_143676) [42]; pch (SAIL_1187_C06) [43]; mlh (SALK_015849) [44]; and msh (SALK_110240) [45] were acquired from Nottingham Arabidopsis Stock Centre. For obtaining zyp double mutants, CRISPR/Cas mediated mutagenesis was performed [46]. Further information is provided in SI Appendix. Coimmunofluorescence was performed on 4% paraformaldehyde preparations [49] or in 3:1 ethanol:acetic acid fixed material followed by antigen recovery [50]. Primary antibodies are the following: rat anti-AtZYP1-C [51], rat/guinea pig antiAtZYP1-C (1:500) [14], rabbit anti-AtZYP1-N (1:500) [38], guinea pig/rat antiASY1 (1:500) [52], rabbit anti-HvHEI10 (1:200) [53], and rat anti-AtSMC3 (1:200). Secondary antibodies at 1:200 are the following: goat anti-rabbit AMCA (Jackson ImmunoResearch), goat anti-guinea pig Alexa Fluor 488 (Abcam), goat anti-rat Alexa Fluor 488 (Invitrogen), and goat anti-rabbit DyLight 594 (Vector laboratories). Super-resolution microscopy methodology is provided in SI Appendix
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